Murine interleukin (IL)-9 inhibits apoptosis in murine T lymphomasvia signal transducer and activator of transcription (STAT)factors. After transfection of the human IL-9 receptor, humanIL-9 had a similar antiapoptotic activity, but, unlike the mouseprotein, inhibited proliferation. This effect was correlatedwith the level of receptor expression and the extent of STATphosphorylation. Expression of a moderate level of suppressorof cytokine signaling 3 (SOCS3) reduced STAT activation by humanIL-9 and prevented inhibition of growth but not of apoptosis.Using mutated IL-9 receptors, we showed that inhibition of proliferationwas correlated with STAT1 and STAT3 activation by IL-9 and inductionof the cell cycle inhibitor p19/ink4d, a STAT3 target gene.Activation of STAT1 by IFN- did not result in cell growth arrest.In this model, cell growth inhibition is therefore associatedwith a higher number of receptors, a more robust STAT activation,and a greater sensitivity to SOCS3 expression, compared to apoptosisinhibition.

IL-94
was initially characterized as a growth factor for T-celllines, mast cell lines, and hematopoietic tumor cells (1)
.However, it is a relatively weak growth factor for normal cellsin vitro. In vivo, IL-9 is involved in immune reactions againstparasites, in asthma, and in lymphoma development (2, 3, 4)
.IL-9 binds to a complex composed of the specific IL-9R and c,a subunit shared with IL-2, IL-4, IL-7, and IL-15 (5)
.

IL-9 signaling involves the activation of JAK1 and JAK3 tyrosinekinases and STAT1, STAT3, and STAT5 transcription factors (5, 6). These factors play a key role in cytokine intracellularsignaling, regulating apoptosis, cell growth, and differentiation(7)
. The mechanism that leads to their activation has beenstudied extensively. STATs are recruited via their SH2 domainto phosphorylated sites of activated cytokine receptors andphosphorylated on a conserved tyrosine by JAK kinases associatedwith the receptor. STAT phosphorylation induces their dimerization,migration to the nucleus, and binding to specific DNA sitesin the promoter of cytokine-regulated genes (8)
. The STAT familycomprises seven distinct members that are implicated in signalingby a large number of cytokines. STAT6 is preferentially activatedby IL-4 and IL-13, whereas STAT4 is activated by IL-12. In contrast,STAT1, STAT3, STAT5A, and STAT5B contribute to the signalingpathways of many factors, including IL-9. In addition, STAT3and STAT5 are constitutively activated in various human lymphomas,leukemias, and breast cancers (9)
.

Both STAT3 and STAT5 are important mediators of apoptosis inhibitionby cytokines, such as IL-6 and IL-9 (10, 11)
. STAT3 and STAT5are activated by a large number of oncogenes and are requiredfor transformation by SRC and BCR/ABL, respectively (12, 13)
.Constitutive activation of these factors in tumors preventsapoptosis (14, 15)
. A mutated STAT3 variant that is constitutivelyactivated has been shown to transform murine cells in vitro(16)
. Development of murine melanoma B16, which presents aconstitutive activation of STAT3, could be blocked in vivo byintroducing DNA coding for a dominant negative form of STAT3into the tumor (14)
.

We had shown that IL-9 and IL-6 protect BW5147 T lymphoma cellsfrom corticoid-induced apoptosis via the activation of eitherSTAT3 or STAT5 (6, 10)
. In the present study, we observedthat IL-9 inhibited the growth of BW5147 cells that expressedhigh numbers of transfected human IL-9Rs. This effect was correlatedwith the number of IL-9Rs present at the cell surface and thekinetics of STAT1 and STAT3 activation.

To determine whether this effect was linked to the level ofreceptor expression, we selected 11 clones expressing the transfectedIL-9R at various levels, as assessed by fluorescence-activatedcell-sorting analysis with a specific anti-hIL-9R monoclonalantibody. The extent of growth inhibition by hIL-9 was alsomeasured for each clone. Fig. 2A
shows that there was a significantcorrelation between the IL-9R level and the reduction of proliferationby hIL-9 (r = 0.83; P = 0.0015). To compare the expression ofthe transfected hIL-9R and the endogenous murine IL-9R, we performeda flow cytometry experiment with fusion proteins consistingof a murine immunoglobulin 3 Fc domain added at the COOH terminusof murine or hIL-9 (27)
. Both chimeric proteins were stillactive. Binding of these proteins was detected by anti-immunoglobulinantibodies conjugated to FITC. The human IL-9-3 construct produceda signal that was about 2-fold and 7-fold stronger than murineIL-9-3 in transfected clones that expressed the hIL-9R at thelowest and the highest levels, respectively (Fig. 2B)
. In bindingstudies using labeled IL-9, we found that cells expressed 1000± 85 murine IL-9Rs/cell and up to 6520 ± 296 transfectedhuman IL-9Rs/cell, confirming flow cytometry data. Taken together,our data suggested that hIL-9R overexpression caused T lymphomagrowth inhibition in the presence of IL-9.

Fig. 2. Growth inhibition by IL-9 is correlated to the level of expression of the transfected receptor. A, IL-9R expression was measured in 11 BW5147 clones by flow cytometry with an anti-IL-9R antibody (6)
. Growth inhibition by hIL-9 was measured as described in Fig. 1
and presented as a ratio between hexosaminidase levels in the absence and presence of IL-9. Linear correlation was calculated using InStat software. B, untransfected cells, clone 6, and clone 45 were stained with a hybrid protein consisting of a constant immunoglobulin 3 domain and either human (solid line) or murine (dashed line) IL-9 and with antimouse immunoglobulin antibodies coupled to FITC. Cells were analyzed by flow cytometry.

Stronger STAT Activation in Cells Overexpressing the IL-9R.
Because murine IL-9 and hIL-9 activate the same set of STATproteins in parental and transfected BW5147 cells, respectively(6)
, we asked whether hIL-9R overexpression could result ina stronger STAT activation, which was tested by Western blottingwith antibodies recognizing the tyrosine-phosphorylated formof STAT1, STAT3, or STAT5. Fig. 3
shows that murine IL-9 induceda transient STAT1 and STAT5 phosphorylation and a more sustainedSTAT3 phosphorylation. Interestingly, phosphorylation of thethree factors was lowest 4 h after stimulation and increasedfrom then on, suggesting the occurrence of STAT activation waves.By contrast, a sustained activation of the three STAT proteinsin response to hIL-9 was observed in the BW-hIL9R cells thatexpressed the higher receptor level (clone 6; Fig. 3
, rightpanel). This was confirmed by electrophoretic mobility shiftassays (data not shown). After 24 h, STAT3 phosphorylation was3-fold more intense, as determined by quantification of theblot presented in Fig. 3
. STAT activation level was intermediatein clone 45, which expressed hIL-9R to a lesser extent and whoseproliferation was only weakly affected by hIL-9. Interestingly,IFN- induced a sustained STAT1 phosphorylation in BW5147 cells,similar to that induced by hIL-9 in BW5147-hIL9R-clone 45 (Fig.3)
but did not affect BW5147 cell growth or enhance growthinhibition by hIL-9 (data not shown). We did not observe phosphorylationof ERK1, ERK2, or protein kinase B kinases in response to murineIL-9 or hIL-9 in these cells, in contrast to other cell lines(28)
.

Fig. 3. Stronger STAT activation in BW5147 cells overexpressing the IL-9R. BW5147 and BW-hIL9R cells were stimulated with murine IL-9 and hIL-9, respectively (500 units/ml), for the indicated periods of time. Alternatively, BW5147 cells were incubated with IFN- (500 units/ml). Cells were lysed directly in sample buffer and loaded (105 cells/well) onto an 8% polyacrylamide gel. Phosphorylation of STAT1, STAT3, or STAT5 was visualized by Western blot with specific antibodies (New England Biolabs), according to the manufacturers instructions. As a control, blots were reprobed with an anti-actin antibody (data not shown). Similar results were obtained in three independent experiments.

Fig. 4. A moderate level of SOCS3 expression blocks growth inhibition but not protection against apoptosis by IL-9. BW5147 cells ectopically expressing a moderate level of SOCS3 (30)
were transfected with hIL-9R in the pEF-myc-cyto plasmid. Clones expressing a similar level of receptor were used. A, phosphorylation of each STAT after IL-9 stimulation was tested by Western blot. B, cells were stimulated with human IL-9 (1000 cells/well). After 3 days, proliferation was evaluated by measuring [3H]thymidine incorporation. C, cells were grown for 24 h with dexamethasone (100 ng/ml) and cyclosporine A (500 ng/ml) in the presence () or absence () of hIL-9 (100 units/ml). The percentage of apoptotic cells was measured by flow cytometry after propidium iodide staining (6)
.

Growth Inhibition Is Correlated with STAT1 and STAT3 Activation and with Induction of p19/ink4d Expression.
We have shown previously that BW5147 growth inhibition dependson a single phosphorylated tyrosine of IL-9R (tyrosine 367 inthe mature protein). Mutation of this amino acid into phenylalanine(mut1) also abolishes apoptosis inhibition and activation ofSTAT transcription factors by IL-9 (6)
. Recently, we describedtwo other mutated IL-9Rs that more specifically activated eitherSTAT5 (mut7) or both STAT1 and STAT3 (mut6) (10)
. Both receptorscould mediate apoptosis inhibition. As shown in Fig. 5
, transfectionof mut7 in BW5147 cells did not affect proliferation, althoughSTAT5 activation in BW-mut7 cells is even stronger than thatin BW-hIL9R cells (10)
. In contrast, mut6 overexpression causedgrowth inhibition by hIL-9, suggesting that STAT1 and/or STAT3were involved. BW-mut6 cells were reproducibly more affectedthan cells expressing the wild-type receptor. This might resultfrom a somewhat stronger activation of STAT1 and STAT3 in BW-mut6because competition with STAT5 at the IL-9R docking site islost in this mutant. In addition, STAT5 activation could havea positive effect on proliferation, which could partially compensategrowth inhibition in BW-hIL9R cells but not in BW-mut6 cells.

Fig. 5. Growth inhibition depends on tyrosine 367 and glutamine 370 of the IL-9R. BW5147 cells were transfected with the following receptors: hIL-9R (wild-type); mut1 (Tyr-367 to Phe, abrogates STAT activation); mut6 (Leu-368 to Arg, results in the preferential activation of STAT1 and STAT3); and mut7 (Gln-370 to Leu, preferential activation of STAT5) (10)
. Clones expressing a similar level of receptor were stimulated with hIL-9. After 3 days, proliferation was evaluated by measuring [3H]thymidine incorporation. A representative experiment is shown, with SDs (bars) calculated from triplicate cultures.

The cell cycle is controlled by cyclin-dependent kinases, whichcan be inhibited by three types of proteins: (a) p21/waf1/cip1;(b) the kip family; and (c) the ink4 family. Several of theseproteins have been implicated in cell growth inhibition by cytokines(17, 20, 22)
. To test whether IL-9 could induce the expressionof such cell cycle inhibitors, we performed reverse transcription-PCRexperiments with oligonucleotides specific for each family member(data not shown). We found that hIL-9 up-regulated ink4d expressionin BW-hIL9R cells, which was confirmed at the protein levelby Western blotting (Fig. 6A)
. p19/ink4d expression was notsignificantly affected by murine IL-9 treatment, suggestingthat this protein could be one of the mediators of hIL-9-inducedcell growth arrest. Moreover, IL-9R mutations that blocked growthinhibition by hIL-9 (mut1 and mut7) also abolished ink4d induction,whereas the mut6 receptor behaved as the wild type (Fig. 6B)
,supporting the role of ink4d as a potential mediator of BW-hIL9Rcell growth inhibition by IL-9.

Fig. 6. Cell growth arrest by IL-9 is correlated with induction of p19/ink4d expression. A, BW5147 and BW-hIL9R cells were stimulated as described above with murine IL-9 and hIL-9, respectively. p19/ink4d expression was tested by Western blot with specific antibodies (Santa Cruz Biotechnology). B, cells expressing the indicated receptor were stimulated for 24 h with IL-9. RNA was extracted and analyzed by Northern blot with a specific 32P-labeled probe. Blots were quantified, and results were presented as a ratio between the signals obtained with the ink4d and the actin probes (C) in the absence (gray columns) or presence (black columns) of IL-9.

In this study, we show that the level of expression of the hIL-9Ris correlated with the intensity of STAT signaling and the abilityof hIL-9 to inhibit cell growth. These results point to thenumber of receptor molecules as a limiting factor for signaltransduction. Accordingly, regulation of cytokine receptor expressionlevels, which is frequently observed, is a potent way of regulatingcytokine responses.

Higher expression of the hIL-9R probably explains the differencebetween murine IL-9 and hIL-9 in regulation of BW5147 cell growth.Overexpression of the IL-9R affected activation of STAT1, STAT3,and STAT5 differentially. STAT1 and STAT5 activation was muchstronger and lasted for a longer time after hIL-9R transfection.STAT3 activation was also increased significantly, althoughit was affected to a lesser extent. STAT3 could have a higheraffinity for the single STAT docking site of the IL-9R and maycompete for binding of other STATs in the presence of limitingnumbers of receptors. Alternatively, there might be some differencesbetween the affinities of the murine IL-9R and the hIL-9R forSTATs, although the IL-9R STAT docking site region is highlyconserved. Activation of phosphatases by these receptors mayalso be different, but there are no available data about activationof such enzymes by IL-9.

Using mutated IL-9Rs, we showed that growth inhibition was correlatedwith activation of STAT1 and STAT3 but not STAT5. STAT1 is agood candidate because its activation is more dramatically affectedby transfection of the hIL-9R. However, because IFN- inducedphosphorylation of STAT1 to a similar extent as hIL-9 in transfectedBW5147 cells but did not inhibit cell proliferation, this activityis unlikely to be mediated by STAT1 alone. In addition, expressionof a dominant negative variant of STAT1 failed to affect thegrowth inhibition effect of IL-9 (data not shown). We observedthat this effect was associated with expression of the cyclin-dependentkinase inhibitor p19/ink4d, a well known inhibitor of cell cycleprogression. This gene has also been implicated in growth inhibitionby IL-6 and IL-10 (22, 31)
. Both studies demonstrated thatink4d induction was mediated by STAT3. Binding sites for thisfactor were found in the ink4d promoter (31)
. Interestingly,in our model, sustained ink4d expression was only observed whenSTAT3 was activated to the highest level. In contrast, a lowerSTAT3 activation by murine IL-6 or IL-9 is sufficient to inducethe expression of various genes, such as Ly-6A/E and Bcl-3 (24, 25). The molecular basis for this difference in sensitivityto STAT3 activation is not clear. Although ink4d up-regulationvia STAT3 most likely plays a role in BW-hIL9R growth inhibition,other genes may also be involved. In this respect, we also observeda modest induction of p21/waf1/cip1 gene expression by hIL-9(data not shown).

In BW5147 cells, STAT3 activation is also involved in apoptosisinhibition in response to IL-6 and IL-9 (10, 30)
. Thus, protectionagainst apoptosis may require a lower level of STAT3 activationthan cell growth inhibition. Our results could explain why STAT3has been shown in some studies to favor tumorigenesis by inhibitingapoptosis and in other reports to block cell growth in similarcell lines (14, 17, 19, 32, 33)
. It is possible that arelatively weak constitutive or induced STAT3 activation isenough to block apoptosis in these cells, whereas a strongeractivation affects cell growth. In line with this hypothesis,tumor cell growth arrest in response to IL-6 often requiresthe addition of soluble IL-6R, which enhances STAT3 activationby this cytokine (17, 33)
. Recently, Nosaka et al.(34)
observedthat transfection of a constitutively activated STAT5 promotedthe growth of Ba/F3 cells, whereas hyperactivation of STAT5by IL-3 in these cells induced differentiation and apoptosis.Similarly, we suggest that the extent of STAT3 activation maycritically affect the outcome for cell proliferation and survival.

Overexpression of SOCS proteins has been shown to completelyblock JAK/STAT signaling by various cytokines, including IL-9(29, 30)
. Here, we show that a moderate level of SOCS3 expression,which is closer to the level that can be achieved in responseto cytokines, has a differential effect on growth inhibitionand apoptosis. This might suggest a more subtle role for SOCSproteins in specifically inhibiting some cytokine effects thatrequire a more sustained signaling intensity.

Besides the JAK/STAT pathway, other mediators might contributeto growth inhibition by IL-9. IRS-1, IRS-2, protein kinase B,and ERK have been involved in IL-9 signaling (28)
. However,we failed to detect phosphorylation of these proteins in stimulatedBW5147 or BW-hIL9R cells. Future studies will have to determinewhether other signaling proteins play a role in this model.

In conclusion, our results suggest that kinetic parameters playa critical role in the regulation of cell growth by the JAK-STATpathway. These parameters are influenced by the level of receptorexpression and the presence of inhibitory proteins such as SOCS3.

1 This work was supported in part by the Belgian Federal Servicefor Scientific, Technical and Cultural Affairs and by the Actionsde Recherche Concertées, Communauté françaisede Belgique, Direction de la recherche scientifique. J-B. D.is a senior research assistant, and J-C. R. is a research associatewith the Fonds National de la Recherche Scientifique, Belgium.